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Background: Antibiotic tagetitoxin inhibits bacterial RNA polymerases (RNAPs) and RNAP III from eukaryotes. Results:We constructed a structural model of tagetitoxin bound to the transcription elongation complex. Conclusion: Tagetitoxin interacts directly with the ␤Ј subunit trigger loop, stabilizing it in an inactive conformation. Significance: Results have implications for designing new antibiotics and understanding principles of RNAP functioning and regulation.

The Id proteins play an important role in proliferation, differentiation and tumorigenesis. Many tumors are hypoxic, but it is unknown if expression of Id proteins is regulated in hypoxic cells. Here we show that Id-1 is down-regulated in multiple primary, immortalized, and neoplastic hypoxic cell lines, and the transcriptional repressor ATF-3 is both necessary and sufficient for this hypoxia-induced repression of Id-1. Hypoxic up-regulation of ATF-3 is due in part to activation of the unfolded protein response, a cellular stress response. Remarkably, we observe that the unfolded protein response is de-regulated in all neuroblastoma cell lines tested. Indeed, in the absence of ATF-3 the hypoxia-induced transcription factor HIF-1 up-regulates Id-1 in hypoxic neuroblastoma cells. Hypoxic neuroblastoma cells diminish expression of some neuronal differentiation markers, and forced expression of ATF-3 in hypoxic neuroblastoma cells represses Id-1 and prevents the loss of these markers. The divergent regulation of Id proteins in distinct hypoxic cells may explain some of the varied effects hypoxia has on cellular differentiation and proliferation.Many tumors are profoundly hypoxic and multiple studies have demonstrated that hypoxic tumors have a poorer prognosis than non-hypoxic tumors. Although the full etiology of this observation is unclear, many of the phenotypes associated with hypoxic cells are due to the induction and suppression of gene expression (reviewed in Ref. 1). While the hypoxia-inducible transcription factor, HIF-1, is the best characterized inducer of gene transcription in hypoxic cells, it is clear that additional signaling pathways can both up-regulate and down-regulate gene expression in hypoxic cells (2, 3). In addition, HIF-1 targets differ dramatically in various cell types despite similar expression of HIF-1␣ (3-6) suggesting that HIF-1 transcriptional activity may be modulated by cell specific factors.The existence of cell-specific factors may play a role in the marked phenotypic differences noted between different hypoxic cell lines and tumors. For example, most normal cells and many neoplastic cells undergo a growth arrest when hypoxic, whereas some stem cells and neoplastic cells continue to proliferate under hypoxic conditions (7,8). Studies have also suggested that hemangioblasts, renal tubular cells, and embryonic stem cells all differentiate when hypoxic, while hypoxic adipocytes and hematopoietic stem cells are resistant to differentiation (9 -12). Neuroblastoma cells, when rendered hypoxic, lose some neuronal markers, leading to the hypothesis that they undergo hypoxic "de-differentiation" to immature neural crest cells (13). Despite the probability that hypoxic regulation of proliferation and differentiation play an important role in the aggressiveness of hypoxic tumors, the mechanisms by which hypoxic cells regulate proliferation and differentiation have not been fully delineated.The Id proteins play an important role in both proliferation and differentiation, although their regula...

BackgroundEmerging resistance of the malaria parasite Plasmodium to current therapies underscores the critical importance of exploring novel strategies for disease eradication. Plasmodium species are obligate intracellular protozoan parasites. They rely on an unusual form of substrate-dependent motility for their migration on and across host-cell membranes and for host cell invasion. This peculiar motility mechanism is driven by the ‘glideosome’, an actin–myosin associated, macromolecular complex anchored to the inner membrane complex of the parasite. Myosin A, actin, aldolase, and thrombospondin-related anonymous protein (TRAP) constitute the molecular core of the glideosome in the sporozoite, the mosquito stage that brings the infection into mammals.MethodsVirtual library screening of a large compound library against the PfAldolase–TRAP complex was used to identify candidate compounds that stabilize and prevent the disassembly of the glideosome. The mechanism of these compounds was confirmed by biochemical, biophysical and parasitological methods.ResultsA novel inhibitory effect on the parasite was achieved by stabilizing a protein–protein interaction within the glideosome components. Compound 24 disrupts the gliding and invasive capabilities of Plasmodium parasites in in vitro parasite assays. A high-resolution, ternary X-ray crystal structure of PfAldolase–TRAP in complex with compound 24 confirms the mode of interaction and serves as a platform for future ligand optimization.ConclusionThis proof-of-concept study presents a novel approach to anti-malarial drug discovery and design. By strengthening a protein–protein interaction within the parasite, an avenue towards inhibiting a previously “undruggable” target is revealed and the motility motor responsible for successful invasion of host cells is rendered inactive. This study provides new insights into the malaria parasite cell invasion machinery and convincingly demonstrates that liver cell invasion is dramatically reduced by 95 % in the presence of the small molecule stabilizer compound 24.Electronic supplementary materialThe online version of this article (doi:10.1186/s12936-015-0834-9) contains supplementary material, which is available to authorized users.

Introduction COVID-19 infection has been hypothesized to precipitate venous and arterial clotting events more frequently than other illnesses. Materials and methods We demonstrate this increased risk of blood clots by comparing rates of venous and arterial clotting events in 4400 hospitalized COVID-19 patients in a large multisite clinical network in the United States examined from April through June of 2020, to patients hospitalized for non-COVID illness and influenza during the same time period and in 2019. Results We demonstrate that COVID-19 increases the risk of venous thrombosis by two-fold compared to the general inpatient population and compared to people with influenza infection. Arterial and venous thrombosis were both common occurrences among patients with COVID-19 infection. Risk factors for thrombosis included male gender, older age, and diabetes. Patients with venous or arterial thrombosis had high rates of admission to the ICU, re-admission to the hospital, and death. Conclusion Given the ongoing scientific discussion about the impact of clotting on COVID-19 disease progression, these results highlight the need to further elucidate the role of anticoagulation in COVID-19 patients, particularly outside the intensive care unit setting. Additionally, concerns regarding clotting and COVID-19 vaccines highlight the importance of addressing the alarmingly high rate of clotting events during actual COVID-19 infection when weighing the risks and benefits of vaccination.

Background The 2011 guidelines for management of pediatric uncomplicated community acquired pneumonia (CAP) recommend the use of ampicillin or penicillin first line. We sought to evaluate improvement in adherence to these guidelines through antimicrobial stewardship interventions at a single institution to minimize unnecessary broad antimicrobials. Methods A retrospective chart review was conducted of admitted patients aged 2 months - 21 years old with uncomplicated CAP. The pre-intervention phase was September 1, 2019-February 29, 2020 and the post-intervention phase included September 1-February 28 of 2021 and 2022. Antimicrobial stewardship interventions included the incorporation of clinical practice guidelines (CPG) into a new institutional CAP pathway (approved in June 2020) and subsequent education to ordering providers in the spring of 2020. Patients with complicated pneumonia or with comorbidities including sickle cell disease, chronic lung disease, neurologic conditions, congenital heart disease or patients who were immunocompromised were excluded. The prescribing patterns of specific physicians were recorded and adherence to CPG recommendations were assessed. The primary endpoint was to measure the reduction of broad spectrum antibiotics (vancomycin, clindamycin, ceftriaxone, levofloxacin and cefdinir) to narrow spectrum antibiotics (ampicillin, amoxicillin, and amoxicillin-clavulanate). Results A total of 114 patients were included in the study; 72 pre-intervention and 42 post-intervention. Mean age was 5.4 years pre- and 6.5 years post-intervention. A significant reduction in broad spectrum antibiotic use was noted in the ED (p=< 0.0001), during the first 24 hours of admission (p=0.0034) and for discharge antibiotics (p=0.0003) (Figures 1-3) between the pre- and post-intervention groups. Guideline adherence was 78.5%. No change in length of stay or treatment failure were observed. Antibiotic Prescriptions in the Emergency Department After the implementation of the CPG guideline there was a 17% reduction in the number of times a combination of antibiotics were used, a 7.2% reduction of broad spectrum antibiotics and a 24% increase in the utilization of narrow spectrum antibiotics, p-value= <0.0001. Antibiotic Utilization 24 hours After Hospital Admission After the implementation of the CPG guideline there was a 20% reduction in the number of times a broad spectrum antibiotic was prescribed, p-value =0.0034. Discharge Antibiotic Prescriptions After the implementation of the CPG guideline there was a 18% reduction of broad spectrum antibiotics and a 34% increase in the utilization of narrow spectrum antibiotics, p=0.0003. Conclusion The CPG and educational interventions had a positive impact on the antibiotic management of children hospitalized, with an overall 4.7% reduction of broad spectrum antibiotics and a 28% increase in the utilization of narrow spectrum antibiotics. Continued education may improve CPG adherence. Disclosures All Authors: No reported disclosures.

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Context: Recommendations for COVID-safe, in-person, high school education have included masks and distancing between students but do not describe a scalable surveillance solution to rapidly identify and mitigate disease prevalence or exposure. Methods: Through an Internet application, all school participants reported symptoms, illness, or exposure daily. Physiciansupervised follow-up interviews were reviewed and recorded in daily rounds. Students and faculty were allowed or prohibited to enter school based on the results. Results: From August 30, 2020, until April 13, 2021, a high school in Bergen County, New Jersey (an epicenter of high COVID prevalence), with 889 students and 214 faculty members, staff, and volunteers, generated 1497 assessments. Reasons for initial evaluation included 48 (3%) participants with positive COVID tests, 520 (34%) COVID-exposed, 178 (12%) exposed to someone with symptoms and unknown COVID status, 208 (14%) subjects with symptoms themselves, 525 (35%) exposed to a high-risk geography or air travel, and 12 (1%) contacts of a contact. Of the 61 subjects ultimately diagnosed with COVID, the sources of infection were 36 (57%) home exposure, 16 (27%) confirmed nonschool sources, 8 (13%) unknown, 1 (2%) travel to a high-risk area, and only one potential case of in-school transmission. Conclusions: Masks, distance, and aggressive contact tracing supported by an Internet application with consistent application of quarantine protocols successfully permitted in-school education without COVID spread in a high prevalence environment. This finding remains important to guide safety measures should vaccine-resistant strains-or new pandemics-challenge us in the future.